Yeast organisms produce a number of proteins that have a function outside the cell. Such proteins are referred to as secreted proteins. These secreted proteins are expressed initially inside the cell in a precursor or a pre-form containing a pre-peptide sequence ensuring effective direction (translocation) of the expressed product across the membrane of the endoplasmic reticulum (ER). The pre-peptide, normally named a signal peptide, is generally cleaved off from the desired product during translocation. Once entered in the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi, the protein can follow different routes that lead to compartments such as the cell vacuole or the cell membrane, or it can be routed out of the cell to be secreted to the external medium (Pfeffer et al. (1987) Ann. Rev. Biochem. 56:829-852).
Insulin is a polypeptide hormone secreted by xcex2-cells of the pancreas and consists of two polypeptide chains, A and B, which are linked by two inter-chain disulphide bridges. Furthermore, the A-chain features one intra-chain disulphide bridge.
The hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acid followed by proinsulin containing 86 amino acids, in the configuration: prepeptide-B-Arg Arg-C-Lys Arg-A, in which C is a connecting peptide of 31 amino acids. Arg-Arg and Lys-Arg are cleavage sites for cleavage of the connecting peptide from the A and B chains.
Three major methods have been used for the production of human insulin in microorganisms. Two involve Escherichia coli, with either the expression of a large fusion protein in the cytoplasm (Frank et al. (1981) in Peptides: Proceedings of the 7th American Peptide Chemistry Symposium (Rich and Gross, eds.), Pierce Chemical Co., Rockford, Ill. pp 729-739), or use a signal peptide to enable secretion into the periplasmic space (Chan et al. (1981) PNAS 78:5401-5404). A third method utilizes Saccharomyces cerevisiae to secrete an insulin precursor into the medium (Thim et al. (1986) PNAS 83:6766-6770). The prior art discloses a limited number of insulin precursors which are expressed in either E. coli or Saccharomyces cerevisiae, vide U.S. Pat. No. 5,962,267, WO 95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741188.
The present invention features novel connecting peptides (mini C-peptides) which confer an increased production yield in insulin precursor molecules and insulin precursor analog molecules when expressed in a transformed microorganism, in particular in yeast. Such insulin precursors or insulin precursor analogs can then be converted into insulin or insulin analogs by one or more suitable, well known conversion steps.
The connecting peptides of the present invention contain at least one Gly and will generally be shorter than the natural human C peptide which, including the flanking dibasic cleavage sites, consists of 35 amino acids. Thus the novel connecting peptides will in general not be of more than 15 amino acid residues in length and preferably not more than 10 amino acid residues. Typically the novel connecting peptides will be of up to 9, 8, 7 or 5 amino acid residues and will preferably not be of more than 4 amino acid residues in length.
As in the natural human insulin molecule, the connecting peptide will contain a cleavage site at its C and N termini enabling in vitro cleavage of the connecting peptide from the A and B chains. Such cleavage sites may be any convenient cleavage sites known in the art, e.g. a Met cleavable by cyanogen bromide; a single basic amino acid residue or a pair of basic amino acid residues (Lys or Arg) cleavable by trypsin or trypsin like proteases; Acromobactor lyticus protease or by a carboxypeptidase protease. The cleavage site enabling cleavage of the connecting peptide from the A-chain is preferably a single basic amino acid residue Lys or Arg, preferably Lys.
Alternatively cleavage of the connecting peptide from the B chain may be enabled by cleavage at the natural LysB29 amino acid residue in the B chain giving rise to a desB30 insulin precursor or desB30 insulin precursor analog. The desired B30 amino acid residue may then be added by well known in vitro, enzymatic procedures.
In one embodiment the connecting peptide will not contain two adjacent basic amino acid residues (Lys,Arg). In this embodiment, cleavage from the A-chain may be accomplished at a single Lys or Arg located at the N-terminal end of the A-chain and the natural Lys in position B29 in the B-chain.
The connecting peptide may comprise more than one Gly but preferably not more than 5. The connecting peptide will preferably not comprise more than 3 Gly and most preferred it will only comprise a single Gly. The Gly may be immediately N-terminal to the cleavage site adjacent to the A chain.
Furthermore, the B27 (atom CG2) will typically have a proximity to the A1 (atom CA) of less than 5 xc3x85.
Accordingly, in one aspect the invention is related to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) being cleavable from the A and B chains said connecting peptide comprising at least one Gly, wherein the B27 (atom CG2) has a proximity to the A1 (atom CA) of less than 5 xc3x85.
In another aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) being cleavable from the A and B chains said connecting peptide comprising at least one Gly and a cleavage site enabling cleavage of the peptide bond between the A-chain and the connecting peptide, wherein one Gly is immediately N-terminal to said cleavage site.
In another aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) being cleavable from the A and B chains said connecting peptide comprising at least one Gly, wherein the connecting peptide is of up to 6 amino acid residues in length.
In a further aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising a sequence of formula:
B(1-27)-X3-X2-X1-Y-A(1-21),
wherein
X1 comprises 1-5 amino acid residues in length comprising at least one Gly,
X2 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain,
X3 is one of Pro, Asp, Lys, or Ile at position 28 of the B chain, and
Y is Lys or Arg.
In one embodiment X1 is 1-4, 1-3 or 1-2 amino acid residues in length.
In a further aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising a sequence of formula:
B(1-27)xe2x80x94X3xe2x80x94X2xe2x80x94X1xe2x80x94Yxe2x80x94A(1-21),
wherein
X1 comprises a Gly immediately N-terminal to Y,
X2 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain,
X3 is one of Pro, Asp, Lys, or Ile at position 28 of the B chain, and
Y is Lys or Arg.
In one embodiment, X1 is 1-15, 1-10, 1-8, 1-5 or 1-3 amino acid residues in length.
In a further aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising a sequence of formula:
B(1-27)xe2x80x94X3xe2x80x94X2xe2x80x94X1xe2x80x94Yxe2x80x94A(1-21),
wherein
X1 comprises at least one Gly,
X2 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain,
X3 is one of Pro, Asp, Lys, or Ile at position 28 of the B chain, and
Y is Lys or Arg,
and wherein the B27 (atom CG2) has a proximity to the A1 (atom CA) of less than 5 xc3x85.
In this embodiment X1 is typically 1-15, 1-10, 1-9, 1-8, 1-5, 1-4 or 1-3 amino length.
In the above formula X1 will typically contain 1-5 Gly, preferably 1-3 and more preferred only one Gly molecule.
The amino acid residues in X1 can be any codable amino acid residue and may be the same or different with the only proviso that at least one amino acid residue in X1 is Gly.
In one embodiment, X3 is Asp and X2 is Lys. This embodiment encompasses the insulin precursor analogs containing an Asp in position B28 of the B chain (termed hereinafter xe2x80x9cAspB28IPxe2x80x9d). In another embodiment X2 is Lys and X3 is Pro. In a further embodiment the sequence X1xe2x80x94Y is selected from the group of: (a) Glu-Glu-Gly-Lys(SEQ ID NO:1, (b) Glu-Gly-Lys, (c) Ser-Gly-Lys, (d) Asn-Gly-Lys, (e) Thr-Gly-Lys, (f) Asp-Gly-Lys, (g) Met-Gly-Lys, (h) Ala-Gly-Lys, (i) His-Gly-Lys and (j) Gly-Lys.
In still further specific embodiments, X1 is 1-3 amino acid residues; X3 is Lys and X2 is Pro. In a further embodiment, X1 is 1-3 amino acid residues, X3 is Asp and X2 is Lys. In another embodiment X2 is Pro, X3 is Lys and X1 is 1-2 amino acid residues of which one is Trp or Phe.
In another embodiment X3 is Lys, X2 is Pro-Thr and X1 consists of up to 15 amino acid residues of which one is Gly. In this embodiment X1 will contain a cleavage site at the C-terminal end, e.g. a mono basic or dibasic (Lys, Arg) cleavage site.
In a specific embodiment, the mini C-peptide comprises the sequence Glu-Gly-Lys, Asn-Gly-Lys, or Asp-Gly-Lys.
In a still further aspect, the present invention is related to insulin precursors comprising a sequence of formula:
B(1-29)xe2x80x94X1xe2x80x94Yxe2x80x94A(1-21),
wherein
X1 is up to 5 amino acid residues in length and Y is a cleavage site.
X1 may be in a further embodiment be of 1-4, 1-3 or 1-2 amino acid residues in length. In another embodiment Y is Lys or Arg. In a further embodiment X1 is GluGly; GluGluGly; SerGly; AsnGly, ThrGly, AspGly; MetGly; AlaGly or HisGly. Thus the sequence X1xe2x80x94Y can be (a) Glu-Glu-Gly-Lys(SEQ ID NO:1, (b) Glu-Gly-Lys, (c) Ser-Gly-Lys, (d) Asn-Gly-Lys, (e) Thr-Gly-Lys, (f) Asp-Gly-Lys, (g) Met-Gly-Lys, (h) Ala-Gly-Lys, or (i) His-Gly-Lys.
The present invention is also related to polynucleotide sequences which code for the claimed insulin precursors or insulin precursor analogs. In a further aspect the present invention is related to vectors containing such polynucleotide sequences and to host cells containing such polynucleotide sequences or vectors.
In another aspect, the invention relates to a process for producing the insulin precursors or insulin precursor analogs in a host cell, said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding the insulin precursors or insulin precursor analogs of the invention under suitable conditions for expression of said precursor or precursor analog; and (ii) isolating the precursor or precursor analog from the culture medium.
In still a further aspect, the invention relates to a process for producing insulin or insulin analogs in a host cell, said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding an insulin precursor or insulin precursor analogs of the invention; (ii) isolating the precursor or precursor analog from the culture medium and (iii) converting the precursor or precursor analog into insulin or an insulin analog by in vitro enzymatic conversion.
In one embodiment of the present invention the host cell is a yeast host cell and in a further embodiment the yeast host cell is selected from to the genus Saccharomyces. In a further embodiment the yeast host cell is selected from the species Saccharomyces cerevisiae. 
In a related aspect, the invention features a mini C-peptide in an insulin precursor or insulin precursor analog wherein the amino acid residues of the C-peptide exhibit sufficient flexibility to allow several geometric arrangements of the C-peptide to accommodate an atomic distance between B27 CG2 and A1 CA less than 5 xc3x85.